2. Academic Validation
  3. Comparison of Classical P81 and SVI-P Cation-Exchange Papers in Radiometric Protein Kinase Assays

Comparison of Classical P81 and SVI-P Cation-Exchange Papers in Radiometric Protein Kinase Assays

  • Biochem J. 2025 Jul 22:BCJ20240731. doi: 10.1042/BCJ20240731.
Muhammad Khabib # 1 2,  Lifang Zhang # 1,  Ashley Ovens 2,  Abdulhameed Al-Ghabkari 3,  John Scott 4 5,  Lindsay Sparrow 1,  Martha Blank 6,  Lisa Murray-Segal 1,  Sandra Galic 1 7,  Morag Park 3 8 9 10,  Christopher Langendorf 2 7,  Naomi Ling 1,  Ashfaqul Hoque 1 7
Affiliations

Affiliations

  • 1 Metabolic Signalling Laboratory, St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia, CA.
  • 2 Protein Engineering in Immunity and Metabolism Unit, St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia, AU.
  • 3 McGill University Rosalind and Morris Goodman Cancer Research Centre, Montreal, Québec, Canada, AU.
  • 4 Monash Institute of Pharmaceutical Sciences Drug Discovery Biology, Parkville, Victoria, Australia, AU.
  • 5 The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia, CA.
  • 6 St Vincent's Institute of Medical Research Bone Cell Biology and Disease Unit, Fitzroy, Victoria, Australia, CA.
  • 7 The University of Melbourne Melbourne Medical School, Melbourne, Victoria, Australia, CA.
  • 8 McGill University Department of Biochemistry, Montreal, Québec, Canada.
  • 9 McGill University Department of Medicine, Montreal, Québec, Canada.
  • 10 McGill University Gerald Bronfman Department of Oncology, Montreal, Québec, Canada.
  • # Contributed equally.
PMID: 40700005 DOI: 10.1042/BCJ20240731
Abstract

Radiometric kinase assays have been widely used due to their high sensitivity and dynamic range. The assay measures the transfer of 32Pi from [γ-32P]-ATP to specific substrates, typically synthetic peptides. The 32P-phosphorylated peptide product is captured by binding it to phosphocellulose paper, specifically P81. Unfortunately, GE Healthcare, the sole supplier of P81, has discontinued its manufacture. Recently, a replacement for P81, SVI-P cation-exchange filter paper, has become available. We have tested SVI-P in various kinase assays, including those for AMPK, Abl, CDK2, and ERK, and found that it performs comparably to P81 in capturing substrates. Additionally, a commercial kinase profiling assay using SVI-P successfully captured a range of peptide and protein substrates from 48 different protein kinases. One minor limitation of SVI-P was the higher background radioactivity; however, this can be addressed through optimisation and extended wash steps. Overall, SVI-P represents a viable alternative for radiometric kinase assays, ensuring continued reliability in both academic and industrial research settings.

Keywords

Biochemical techniques and resources; P81; SVI-P; [γ-32P]-ATP; kinases; radiometric kinase assay.

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